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CNAG Sample Requirements
Code: INS-001
Rev: A
Page: 1 of 9
General considerations:
-
Sample and shipment details for DNA and RNA samples should be discussed with the
CNAG BIorepository (Lídia Agueda, Head of Biorepository, [email protected]).
-
Sample and shipment details for prepared libraries and ChIP experiments should be
discussed with Sample Preparation team Manager (Julie Blanc, [email protected]).
-
CNAG BIorepository can provide advice on DNA/RNA extraction methods if extraction
has not been performed yet.
-
When project details are set up (number and type of samples) the CNAG Biorepository
will provide the collaborator with appropriate sample tubes and unique barcodes for
sample shipment and identification. The provided labels contain: project ID, unique
barcode and sample type. The use of these tubes and labels is mandatory.
-
DNA and RNA sample types can be classified into:
Genomic DNA
Cloned DNA
DNA
PCR amplicons
cDNA
Total RNA
RNA
polyA RNA (mRNA)
rRNA-depleted RNA
-
Additional tubes and labels can be requested from the CNAG Biorepository, never use
different tubes or non CNAG- labels for tube identification
-
.
Before sample shipment the total amount of required material needs to be discussed
with CNAG Project Manager as it may vary in function of the experiment type and/or
the genome size of the organism. Most commonly used protocols and sample
requested minimal quantities are:
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
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Code: INS-001
Rev: A
Page: 2 of 9
CNAG Sample Requirements
Protocol
Minimal quantity requested
(fluorescence based quantification
method)
Whole Genome Sequencing without PCR
5 ug
Low Input Whole Genome Sequencing with PCR
300 ng*
Low Input Whole Genome Sequencing without PCR
500 ng*
Bisulphite Whole Genome Sequencing
5 ug
Mate Pair Sequencing
6 ug (1 library)
RNA Sequencing
2 ug of total RNA;
400 ng of rRNA depleted RNA
dirRNA Sequencing
2 ug of total RNA;
400 ng of rRNA depleted RNA
Custom and Exome Capture Sequencing:
Agilent SureSelect
6.5 ug
Custom and Exome Capture Sequencing:
Nimblegen SeqCap
2.5 ug
Genotyping by Sequencing
2.5 ug
*Contact CNAG Project Manager for further details.
Collaborator Sample Data File for DNA and RNA samples:
-
CNAG Biorepository will provide the Collaborator Sample Data File (FOR-002) to be
filled in by the collaborator (in Excel format).
-
FOR-002 file should provide the entire sample information for appropriate
identification and posterior analysis. The correct completion of this document is
crucial for the success of the project. Please do not modify anyhow the Excel, do not
delete or add any columns (the information is automatically uploaded into the CNAG
LIMS).
-
Please, complete the FOR-002 English.
-
FOR-002 verification by 2 independent persons is highly recommended as any error at
this stage will be detrimental to the project.
-
Sending of replacements or additional material:
a) if any sample needs to be replaced by a NEW ONE (new individual), use a new
barcode for sample identification and fill in FOR-002 with its same sample_name
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
Written by:
Review by:
Approved by:
Date:
CNAG Sample Requirements
Code: INS-001
Rev: A
Page: 3 of 9
(that should be different from the sample being replaced). Also is recommended
to fill in comments column with “to sequence instead of sample_barcode of the
replaced sample”
b) if additional material FROM SAME SAMPLE needs to be send, use a new barcode
for sample identification, fill in FOR-002 with exactly the same sample_name and
fill in the column “replacement_of” with the barcode of the sample that has been
re-send.
-
FOR-002 must be sent by e-mail to CNAG Biorepository BEFORE sample shipment
([email protected]).
-
Contact CNAG Biorepository for any questions or doubts regarding the FOR-002
document ([email protected]).
The Collaborator Sample Data File (FOR-002) contains the following fields:
All fields are mandatory except for those labeled “Optional”.
Column
1
Field name
PROJECT_CODE
2
LAB_CENTER
3
COHORT_NAME
4
SAMPLE_BARCODE
5
REPLACEMENT_OF
6
SAMPLE_NAME
Field description:
Acronym for the Project identification.
Provided by CNAG.
Optional.
Laboratory identifier.
Use only alphanumerical characters (no spaces, dashes or
dots).
Optional.
Cohort identifier.
Use only alphanumerical characters (no spaces, dashes or
dots).
Aliquot unique identifier.
Provided by CNAG in the same order as printed labels.
Check that barcodes in the file correspond to ship barcode
labels.
Sample barcode of the original sample that is being
replaced by this new aliquot.
When a sample is a replacement of a previous one,
SAMPLE_NAME must be exactly the same (see page 2).
Sample unique identifier.
No human name or surname should appear in the file.
Two aliquots from same sample must have same
SAMPLE_NAME but different SAMPLE_BARCODE.
When additional material from the same sample is
required, both samples must have same
SAMPLE_NAME but different SAMPLE_BARCODE.
If there are different samples from same individual
(p.e. normal/tumor; treated/untreated…) those
must have different SAMPLE_NAME.
Two or more experimental replicates that need to
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Berta Fusté
Marta Gut
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CNAG Sample Requirements
7
SAMPLE_ TYPE
8
SPECIES
9
MATERIAL_SOURCE
10
EXTRACTION_METHOD
11
RESUSPENSION_BUFFER
12
INITIAL_VOLUME
13
STOCK_CONCENTRATION
13
15
ABSORBANCE_RATIO_
260/280
ABSORBANCE_RATIO_
260/230
SEX
16
STATUS
17
PEDIGREE
18
FATHER
14
Code: INS-001
Rev: A
Page: 4 of 9
be all sequenced must have different
SAMPLE_NAME and different SAMPLE_BARCODE.
Use only alphanumerical characters (no spaces, dashes or
dots).
Type of material
(g DNA , total RNA, small RNA…)
Provided by CNAG. Discuss with CNAG staff available
possibilities.
Specify the species from which the DNA/RNA has been
obtained.
Use NCBI Taxonomy Browser compatible species names.
For non-human samples, add known/approximate genome
size in the COMMENTS column.
Specify the source (e.g. whole blood, buccal swabs, FFPE,
liver, whole organism, etc.) from which the DNA/RNA has
been obtained.
Nucleic Acid extraction method employed.
Please specify kit and manufacturer, if known.
Buffer used in final resuspension for the material
obtention.
Sample volume in µl.
Exact volume provided to CNAG, no
approximations.
Sample concentration in ng/µl.
Accepted concentration may vary according to the
project characteristics, please, discuss with CNAG
staff.
In quantification method employed is different
than picogreen or equivalent; please specify it in
COMMENTS column.
ABSORBANCE RATIO 260/280
If samples are quantified by absorbance methods.
ABSORBANCE RATIO 260/230
If samples are quantified by absorbance methods.
Sex of the individual (coded).
0=unknown or not applicable; 1= Male; 2= Female.
Mandatory.
Status of the individual (coded).
0 = unknown or not applicable, 1 = unaffected/normal/
control/wild type, 2 = affected/tumor/index case.
Mandatory.
Pedigree identifier.
Members of same family will have same PEDIGREE
identifier. See example below.
Optional, mandatory for family studies.
Sample_name of the father of this individual.
Must be coincident (exact same spelling) with the
SAMPLE_NAME of the father. See example below.
Optional, mandatory for family studies.
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
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CNAG Sample Requirements
19
MOTHER
20
GEOGRAPHIC_ORIGIN
21
COMMENTS
Code: INS-001
Rev: A
Page: 5 of 9
Sample_name of the mother of this individual.
Must be coincident (exact same spelling) with the
SAMPLE_NAME of the mother. See example below.
Optional, mandatory for family studies.
Geographic origin of the sample.
Mandatory.
Any comments that the collaborator wishes to add, and/or
any of the previously mentioned:
For non-human samples, add known/approximate
genome size.
Quantification method (if different than
Nanodrop).
Optional.
Sample shipment:
-
Always notify to the CNAG Biorepository staff about shipments BEFORE they are sent
([email protected]) .
-
It is imperative that all sample tubes are sent in the same parcel.
-
If possible, provide shipment tracking information upon shipment.
-
Shipment time:
Send parcels preferably at the beginning of the week.
Receipt of parcels later than 16h Monday to Thursday and 12 noon on
Fridays is not possible. There is no delivery on Saturdays or Sundays. CNAG
will not be responsible for parcels received outside of these time frames.
Please, confirm with CNAG Biorepository staff the reception timetables during
bank holidays dates and summer or Christmas period. Bank holidays differ
between regions and Countries.
-
Shipment address:
ATT. Lídia Agueda, PhD
Centre Nacional de Anàlisi Genòmica (CNAG)
Parc Científic de Barcelona
C/Baldiri i Reixac, 4. Torre I, 1era planta
Barcelona 08028 – Spain
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
Written by:
Review by:
Approved by:
Date:
Code: INS-001
Rev: A
Page: 6 of 9
CNAG Sample Requirements
DNA sample requirements:
-
The amount of total material required depends on the study and needs to be agreed
with CNAG staff before the shipment of the samples.
-
The DNA must be pure and intact, of high molecular weight and free of contaminating
nucleic acids from other individuals or other species.
-
The DNA must not be PCR amplified (no Whole Genome Amplified samples).
-
DNA must be free of RNA. Depending on the extraction method, RNAse treatment will
be needed.
-
The sample buffer must be 10mM Tris/1mM EDTA or water.
-
DNA should be quantified using a double stranded DNA specific method such as
Picogreen (or equivalent florescent-based quantification method). We consider OD
quantification inadequate (ie. Nanodrop).
-
DNA must be given at a concentration between 50 and 200ng/ul
-
Samples outside of this concentration range (50-200ng/ul) cannot be accepted and will
.
be returned.
-
If OD quantification is the only option available, please indicate this in comments
column and provide absorbance ratios 260/280 and 260/230. OD 260/280 must be
between 1.8 and 2.0 and OD 260/230 between 1.8-2.2.
-
DNA samples can be sent at room temperature or refrigerated (~4ºC).
At CNAG DNA Quality Control consists of integrity check on agarose gel electrophoresis,
detection of present incidental inhibitors by regular PCR amplification and fluorescence-based
quantification.
RNA sample requirements:
-
The amount of total material required depends on the study and needs to be agreed
with CNAG staff before the shipment of the samples.
-
RNA must be free of DNA and must have good integrity (RIN >8)
-
The sample buffer must be water.
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
Written by:
Review by:
Approved by:
Date:
CNAG Sample Requirements
-
Code: INS-001
Rev: A
Page: 7 of 9
RNA must be given at normalized concentration between 50-200ng/ul for totalRNA
samples and between 10-50 for rRNA depleted RNA samples
. We strongly
recommend florescent-based quantification methods. If concentration values are
measured by spectrophotometric measures (e.g. Nanodrop) please indicate this in
comments column and provide absorbance ratios 260/280 and 260/230.
-
RNA samples must be sent frozen on dry ice.
-
At CNAG RNA samples Quality Control consists of fluorescent-based quantification
(Ribogreen) and integrity evaluation (Agilent 2100 Bioanalyzer).
If CNAG determines in its sole discretion that any of the samples do not meet the criteria set
forth in the previous description, CNAG will so inform the Collaborator. At such time,
Collaborator will be provided the option (a) replace such samples, or (b) proceed with such
samples. In both (a) and (b), Collaborator shall pay CNAG full price for the sequencing of all
samples, even if no successful results are obtained for those samples.
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
Written by:
Review by:
Approved by:
Date:
CNAG Sample Requirements
Code: INS-001
Rev: A
Page: 8 of 9
All matters regarding DNA/RNA sample transfer to CNAG please contact:
Lídia Agueda, PhD
Head of Biorepository
Tel:+34 934020569
E-mail: [email protected]
All matters regarding project design please contact:
Berta Fusté, PhD
Project Manager
E-mail: [email protected]
All matters regarding prepared libraries or partially prepared libraries
Please contact:
Marta Gut, PhD
Head of Sequencing
E-mail: [email protected]
Julie Blanc, MSc
Sample Preparation Manager
E-mail: [email protected]
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
Written by:
Review by:
Approved by:
Date:
CNAG Sample Requirements
Code: INS-001
Rev: A
Page: 9 of 9
Example of collaborator sample data file (FOR-002):
PROJECT_CO LAB_CENTER COHORT_ SAMPLE_ REPLACEMENT SAMPLE_ SAMPLE_ SPECIES
(opt)
NAME
BARCODE _OF
NAME
TYPE
DE
(opt)
MATERIAL_ EXTRACTION RESUSPENSION_ INITIAL_ STOCK_CONC absorbance absorbance SEX
SOURCE
_METHOD BUFFER
VOLUME ENTRATION _RATIO_
_RATIO_
(opt)
(ul)
(ng/ul)
260/280
260/230
EXAMP
EXAMP
EXAMP
EXAMP
blood
blood
blood
blood
PCB
PCB
PCB
PCB
K035
K036
K037
K038
id1
id2
id3
id4
gDNA
gDNA
gDNA
gDNA
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
salting out
salting out
salting out
salting out
water
water
water
water
100
120
100
110
210
212
189
200
1,8
1,8
1,8
1,8
2,2
2,2
2,2
2,2
STATUS PEDIGREE_ FATHER MOTHER GEOGRAPHIC COMMENTS (opt)
(opt)
NUMBER
(opt)
(opt)
_ORIGIN
(opt)
(opt)
1
2
2
1
“id1” is the father of “id 4”
Lídia Agueda
Berta Fusté
Marta Gut
29/05/2014
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1
1
1
2
322
322
322
322 id1
id2
Barcelona
Barcelona
Barcelona
Barcelona
picogreen quantification
picogreen quantification
picogreen quantification
picogreen quantification
“id2” is the mother of “id 4”